Cell lines

ABSTRACT

Genomic instability in T-antigen expressing cells can be overcome by modifying the gene expressing T-antigen so that it lacks Bub1 binding. Stable cell lines can be produced by incorporation of the modified T-antigen gene, preferably together with the catalytic sub-unit of the telomerase construct.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.11/238,702, filed Sep. 29, 2005, which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

FIELD OF THE INVENTION

The present invention relates to the production of cell lines useful intherapy, in particular for transplantation for the treatment ofneurological disorders, including stroke, Huntington's disease,Alzheimer's disease, Creutzfeld-Jacob disease and traumatic braininjury.

BACKGROUND TO THE INVENTION

Stroke is the name given to sudden neurological deficits most commonlycaused by obstruction or haemorrhage of an artery supplying a region ofthe brain. Any region of the brain can be affected.

There are an estimated twelve million survivors of stroke in theprincipal European, American and Japanese markets, with the number ofnew cases in these markets growing at 7 percent per annum. Approximately30 percent of stroke patients require ongoing nursing care, estimated tocost between $25 billion to $30 billion per annum in the US alone.

The 20 percent of survivors with moderate or severe disability afterstroke are potential candidates for neural stem cell transplantationtherapy. Existing drug treatments serving these patients are limited andseek to address the effects rather than the cause of the condition.

Huntington's disease is an uncommon, inherited, progressive and fatalneurodegenerative disorder. In the US, approximately 35,000 patientsshow overt signs of the disease, with a further 75,000 carrying theabnormal gene. There are no existing treatments for the disease.

Dementia is the most devastating and costly age-related disorder. Themost frequently encountered type is senile dementia of the Alzheimer'stype (AD), although some (but not all) treatments for Alzheimer's mayeventually prove applicable to other dementias. Currently, the cost ofcaring for Alzheimer's patients, who can no longer safely care forthemselves, is estimated to approach $100 billion in the US. Age itselfis a risk factor for Alzheimer's, the prevalence of Alzheimer's in thepopulation doubles from age 65 to 75, and again from 75 to 85, at whichpoint 35% show signs of AD. With the ageing of the population, over thenext two decades the number of Alzheimer's patients in the US alone willincrease from 4-5 million to ten million cases. The social and financialburdens will expand proportionally. Dementia is also seen following lossof blood supply to the brain, for example, following cardiac arrest orsome forms of cardiac bypass surgery, also following suffocation orfollowing multiple infarcts in the brain.

Creutzfeld-Jacob disease is a rare disorder, one of a number oftransmissible spongiform encephalopathies involving progressiveinflammatory neurodegeneration occurring due to the buildup ofphysicochemically abnormal prion protein. A variant form is seen incases of variant CJD, a rapidly progressing fatal prion diseasemirroring the bovine disorder, “mad cow” disease or bovine spongiformencephalopathy (BSE). The theoretical risk of an epidemic of thisdisorder in the UK and possibly elsewhere remains, due to the widespreadconsumption of meat products from BSE-infected cattle during the 1980sand early 1990s.

Traumatic Brain Injury significantly affects over 500,000 individualsannually in the United States alone with a variety of affects from mildconcussion to coma and death. 80-85,000 patients annually suffer a headinjury that leads to very significant neurological deficit ordisability. The estimates of total economic cost place the price tag forTBI at almost $50 billion in the US alone.

Stem cell replacement therapy is seen as a viable treatment option formany diseases including, but not limited to, those described above forwhich significant cell loss and damage is a cause or consequence. Stemcells can be derived from human tissues at any stage of development,from the early embryo to the adult. Early embryonic stem cells arecapable of forming cells from any tissues; however, the cells are likelyto form tumours when transplanted. As the tissues develop through thefetal and adult stages, the resident stem cell populations reduce theirdevelopmental potential and lose their inherent tumourigenic capacity,becoming somatic stem cells, also known as tissue- or lineage-restrictedstem cells. Somatic stem cells are multipotent, that is they are capableof becoming any differentiated cell type from their organ of origin.

Embryonic stem cells can be ‘differentiated’ or selected to formpopulations of somatic stem cells, which phenotypically resemble somaticstem cells from fetal or adult tissues and therefore lose theirtumorigenic potential (for example, WO03-A-000868). Only somatic stemcells (derived from adult or fetal tissues or differentiated fromembryonic stem cells) represent, at the current state of knowledge, safestem cells for cell therapy. The cell lines described in this inventionare examples of somatic neural stem cells.

Somatic stem cells from the brain have been proposed as treatments forintractable neurological disorders including Parkinson's diseases,stroke, Huntington's, and and spinal cord injury. Despite early successin anecdotal clinical reports, controlled transplant studies withprimary human fetal brain tissue in Parkinson's disease have, however,failed to deliver any consistent benefits [Freed CR, et al., NEW ENGLANDJOURNAL OF MEDICINE 344 (10): 710-719 MAR. 8, 2001; Olanow et al.,ANNALS OF NEUROLOGY 54 (3): 403-414 SEPTEMBER 2003] and widespreadclinical application of such transplants is in any case constrained bypractical and ethical problems.

The heterogeneity of primary human fetal tissues and cells in terms ofboth cell purity and quality makes the interpretation of such clinicaltrials difficult. Alternative products can be built on new knowledge ofthe potential of stem cells as a source of scalable purified cells andtissues for transplantation. For example, following the demonstrationthat neuroepithelial stem cells and, significantly, clonal cell linesderived from neroepithelial stem cell populations, can restorefunctional deficits in animal models of neurological diseases [Sinden etal., NEUROSCIENCE 81 (3): 599-608 DECEMBER 1997; WO-A-9710329; Gray etal., PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIESB-BIOLOGICAL SCIENCES 354 (1388): 1407-1421 AUG. 29, 1999], many groupshave shown that fetal human neural cells can be expanded for severalmonths in defined media with additional growth factors either asgenetically immortalized (by means of the transduction of differentimmortalizing genes) or as expanded stem cells using specialised culturemethods (sometimes referred to as ‘epigenetic’ methods) [Flax et al.,NATURE BIOTECHNOLOGY 16 (11): 1033-1039 NOVEMBER 1998; Vescovi et al.,EXPERIMENTAL NEUROLOGY 156 (1): 71-83 MARCH 1999]. Neural stem cells, asdescribed in the prior art, have been transplanted into experimentalanimals and have shown evidence of survival. However, these cells andcell lines are not suitable for use in human patients due to theiruncontrolled provenance and manufacture and have not as yet shownevidence of functional efficacy in validated animal models of humanneurological disease.

Real clinical and industrial progress in human stem cell transplantationis dependent upon the availability of cell lines with controlled sourcesand manufacturing that are able to expand quickly and serve as asustainable resource, available on demand to a broad population ofpatients. Cell lines could be specific to individual disorders orgeneral across a class of disorders. In order to achieve this goal, celllines must be generated with appropriate biological characteristics(tissue or cell specific phenotype) and must be sufficiently robust tosurvive a scaleable manufacturing process to make master and workingcell banks of frozen vials of cells from which reproducible,GMP—compliant, clinical lots and commercially viable product batches canbe derived. One approach to this problem is to use an immortalizing geneto safeguard the regenerative potential of a cell line and prevent itfrom entering early senescence. Examples of immortalising genes thathave been used to generate neural stem cell lines include: (1)telomerase reverse transcriptase (hTERT) [Telomerase immortalization ofneuronally restricted progenitor cells derived from the human fetalspinal cord Roy N S, Nakano T, Keyoung H M, Windrem M, Rashbaum W K,Alonso M L, Kang J, Peng W G, Carpenter M K, Lin J, Nedergaard M,Goldman S A NATURE BIOTECHNOLOGY 22 (3): 297-305 MARCH 2004], (2) SV40 Tantigen [Sinden et al., op cit, WO9710329], (3) combinations of SV40 Tand hTERT (WO0121790) and myc proteins [U.S. Pat. No. 5,580,777; Flax etal., op cif]. Genetic overexpression of c-myc, a naturally occurringprotooncogene that is normally expressed during cell development, isdemonstrated in this invention as a means of stably enhancing cellproliferation and preventing changes in cell karyotype, thereby avoidingtransformation of the cell phenotype.

Following from considerable experience in developing stem cell lines astherapeutics, we believe the following represent the key features of ahuman stem cell line for application in the clinic:

-   -   Multipotent cells that can develop into specific cells typical        of the tissues that are targeted for transplantation.    -   Cells that are derived from a single founder cell (clonal cells)    -   Genetically stable cells with normal chromosomes    -   Cells that have the ability to differentiate into appropriate        cell types, in vitro and in vivo.    -   Cells that can be grown in large numbers and stored    -   Cells that are safe, particularly not showing tumourigenic        potential    -   Cells whose migration, once implanted, is limited to areas of        tissue damage    -   Cells that are efficacious in recognised animal models    -   Cells whose provenance is fully documented

While expansion of stem cells on surface or suspension culture ispossible without genetic immortalisation (‘epigentic’ culture),expansion of human neural stem cells is slow and the resulting culturescontain a high proportion of differentiated progeny, which will notsurvive subculture manipulations [WO99-A-11758, Vescovi et al., op cit].Further, long term culture of ‘epigenetic’ stem cells may inducechromosomal alterations that may prevent the clinical application of thecell lines. There are no reports of genetic stability of humanepigenetic somatic cell lines, although recent reports have indicatedchromosomal aberrations in embryonic stem cell lines at greater passagenumbers [Draper et al., NATURE BIOTECHNOLOGY 22 (1): 53-54 JANUARY 2004;Inzunza et al., MOLECULAR HUMAN REPRODUCTION 10 (6): 461-466 JUN. 12004].

Therefore, the scalability of epigenetically expanded cell lines islimited from an industrial point of view and the product may vary fromcell passage to cell passage. The use of an immortalising gene that willnot generate a transformed cell phenotype or otherwise influence thestem cell's biological potency or safety but otherwise enhance thecell's industrial scalability and stabilise the karyotype is highlydesirable.

Over the past 10 years increasing information has been emerging aboutthe role of myc oncogenes in normal and cell proliferation,differentiation and apoptosis. To maintain cell proliferation Myc andMax proteins dimerise and translocate to the cell nucleus where theyserve as transcription factors. Recently Coller et al., [PROCEEDINGS OFTHE NATIONAL ACADEMY OF SCIENCES, 2000; 97:3260-3265] identified 27genes that were induced, and 9 genes that were repressed by activationof c-myc (cellular myc) in primary human blastocysts. Induced targetsincluded cell cycle genes (e.g. G1 cyclin D2) required to maintaindivision, and pro-apoptotic genes (e.g. TRAP1) involved in cell death,while repressed targets included genes encoding extra cellular matrixand cytoskeletal proteins, indicating a role for myc in cell adhesionand structure. A recent report has shown that a v-myc (viralmyc)-immortalised murine neural stem cell line is able to promote theregeneration of damaged neurons in a Parkinson's disease animal modeland promote functional recovery [Ourednik et al., NATURE BIOTECHNOLOGY20 (11): 1103-1110 NOVEMBER 2002].

The ability of c-myc to maintain cell proliferation makes it a primecandidate for stem cell immortalisation, provided that cell division canbe regulated in vivo. For therapeutic use of myc-immortalised cells, apreferred embodiment would permit control over the function of the Mycprotein, such that the immortalising protein was not functional aftertransplanting the cells. This would reduce the risk of overgrowth ortumour formation by the transplanted cells. A preferred conditional formof Myc is a fusion between Myc and the hormone-binding domain of amodified estrogen receptor [Littlewood, et al., NUCLEIC ACIDS RESEARCH,1995; 23, 1686-1690].

SUMMARY OF THE INVENTION

The present invention is based on the development of cell lines thathave favourable characteristics making them useful in transplantationtherapy.

According to a first aspect of the present invention, an isolated cellis obtainable from any of the cell lines having the ECACC Accession Nos.04091601, 04092302 and 04110301.

According to a second aspect of the present invention, a cell identifiedabove, is used in therapy.

According to a third aspect of the present invention, a cell identifiedabove is used in the manufacture of a medicament for the treatment of adisorder associated with loss of our damage to brain cells.

We have generated neural stem cell lines using the regulateable mycimmortalising gene, modified to produce a fusion protein of Myc and thehormone binding domain of a modified estrogen receptor. The fusionprotein is selectively activated by the synthetic hormone 4-hydroxytamoxifen (4-OHT). Using a regulateable immortalising gene allows us tomaximise conditions for unrestricted cell growth and expansion inculture whilst enabling the cells to terminally differentiate in theabsence of 4-OHT when transplanted.

DESCRIPTION OF THE DRAWINGS

The invention is illustrated with reference to the accompanying drawingswherein:

FIG. 1 is a graph showing the growth characteristics of the cell linedesignated CTXOE03;

FIG. 2 is a graph showing cell proliferation of the cell line CTXOE03supplemented with growth factors; and

FIG. 3 is a graph showing the growth characteristics of the cell linedesignated HPC-OA07

DESCRIPTION OF THE INVENTION

The present invention discloses the preparation of cells that aresuitable for transplantation therapy and which are immortal up to thetime of transplantation.

The cells are modified with the incorporation of a conditional form ofMYC oncogene which is controlled by the oestrogen receptor.

The recombinant cells of the invention have use in therapy. Inparticular, the cells of the invention may be used in the treatment ofbrain damage. The brain damage may be caused by a degenerative diseaseor by trauma or hypoxia. In a preferred embodiment, the cells are usedin the treatment of Huntington's disease or Alzheimer's disease.

The cells of the invention may be used in a screening assay to identifybiological or chemical agents that are of potential use in celldifferentiation or which play a role in neural cell development.Screening assays may also be performed to identify agents that have apossible beneficial effect in the treatment of a neurological disease ordisorder. The assays are performed by contacting the agent with aculture of one or more of the cells of the invention, and determiningwhether the agent has an effect on the cell. The assays may be performedin suspension culture, or with adherent cells attached to a substratesurface. The effect may be the ability of the agent to influencedifferentiation of the cells or the cells' growth characteristics.Alternatively, the agent may be a potential drug and the screening assayis performed to study the toxicology of the agent on the cells.

Methods for the preparation of formulations for delivery to a patientwill be apparent to the skilled person. Suitable excipients, diluentsetc., will again be apparent based on current practice in preparingcell-based therapies. The amount of cells required for delivery willvary depending on the form of treatment, the severity of thedisease/damage, and the need for applying multiple doses over atreatment period. However, the skilled person can readily determine theappropriate treatment based on existing cell transplantation therapies.

The cell lines will now be described in further detail. The cell lineshave been deposited at the European Collection of Animal Cultures,Vaccine Research and Production Laboratories, Public Health LaboratoryServices, Porton Down, Salisbury, Wiltshire, SP40 JG, UK. The Accessionnumbers and dates of deposit are as follows:

Cell Line Deposit Date ECACC Accession Number CTX-OE03 Sep. 16, 200404091601 STROC05 Nov. 4, 2004 04110301 HPCOA07 Sep. 23, 2004 04092302

(1) Derivation and Provenance of the Cell Line CTX0E03

Cell Line Summary

A c-MycER^(TAM) transduced human neural stem cell line was derived from12 week fetal cortex. The line was maintained on laminin coated cultureflasks using defined serum-free “Reduced Modified Media” (composition tobe described below) in the presence of bFGF, EGF and 4-hydroxytamoxifen. In routine culture the cell line has a doubling time ofaround 2-3 days.

In growth medium the cells are nestin positive beta-III tubulin negativewith a low percentage of GFAP positive cells. Following differentiationfor 7 days there is up-regulation of beta III tubulin expression andacquisition of a neuronal morphology. A low level background of GFAPexpression is maintained following differentiation.

Following differentiation there is no expression of MHC class I or classII antigens.

Molecular phenotyping by RT-PCR has confirmed the cells to be mycERpositive and nestin positive. In addition a range of neural developmentgenes have been identified as expressed.

This cell line is genetically normal, male XY karyotype, at passage 31(over 100 population doublings). The cell line is clonal by Southernblot and the genome integration site has been identified withinchromosome 13. No known genes are disrupted by this insertion.

Maternal serology indicates the donor to be free from adventitiousinfections, although a history of prior cytomegalovirus infection wasfound (CMV ab IgG positive, IgM negative).

pLNC-mvc-ER^(TAM)

In order to generate a myc-ER driven by a strong promoter themycER^(TAM) transgene was further cloned into the retroviral vectorpLNCX-2 (clontech) which contains a cytomegalovirus promoter (CMV) and aneomycine resistance gene. Plasmid DNA pBabe-puro-myc-ER^(TAM) wasamplified as above. Myc-ER^(TAM) sequence in pBabe-puro-myc-ER^(TAM) wasexcised by restriction enzyme EcoR I. A 2.3 kb fragment of myc-ER^(TAM)was isolated and ligated to Stu I site of pLNCX-2 retroviral vector byblunt-end ligation to generate pLNC-myc-ER^(TAM). The orientation ofmyc-ERTAM gene in pLNC-myc-ER^(TAM) is determined by restrictiondigestion with BamH I, Xho I and Bgl II Stock plasmid DNApLNC-myc-ER^(TAM) was amplified by “Maxi-Prep”.

Generation of TEFLY-A Producer Lines

The TEFLY-A and TEFLY-RD virus packaging cell lines (Obtained underlicence from CRC UK; U.S. Pat. No. 6,165,715; GMP Cell Bank stocks heldby Genethon, Evry, France) were used to produce MMLV based retroviruses.The cell lines retain the gag, pol and env, genes whereas the viralgenome is replaced by the engineered transgene of choice in a retroviralplasmid. TEFLY-RD is used to package virus with restricted infectivityincluding the TEFLY-A lines. TEFLY-A is used to package/generateamphotropic virus with broad spectrum infectivity for mammalian cells.

pLNC-mvcER^(TAM) Virus

TEFLY-RD producer cells were revived from frozen stocks and new seedstocks established. From expanded stocks, 1.5 million cells were seededinto a 10 cm dish and transfected with 12 μg of pLNC-mycER^(TAM) plasmidusing Fugene-6 by standard procedures. Fresh TE-media was put on thetransfected cells overnight and packaged virus was harvested from thecells the next morning. This transient virus production was used toinfect TEFLY-A producer cells.

Newly acquired TEFLY-A producer cells (TEFLY-A Lot 97-8-02A) wererevived from frozen stock and seed stocks established. From the seedstocks, cells were revived, plated out, and infected with supernatantfrom TEFLY-RD containing pLNC-mycER^(TAM) packaged virus, in thepresence of 8 μg/ml polybrene. Infected TEFLY-A producer cells wereexpanded in culture for 2-3 weeks in the presence of neomycin(G418/Geneticin) to generate a selected (by antibiotic resistance) bulkpopulation of mycER^(TAM) virus producer cells. This bulk population wasplated at low density to isolate individual clones. Sixty four cloneswere isolated by ring cloning and passaged into 24 well plate (1clone/well). Individual TEFLY-A clones were expanded up to T25 cultureflasks at which point cells were prepared for viral harvest over 8 h infresh TE media. Virus was titred against Te671 cells by serial dilutionof the collected media in the presence of 8 μg polybrene. A clone withapparent titres of >10⁶ cfu/ml was expanded to generate a working stockof 40×1 ml aliquots of 5×10⁶ cells. Viral stocks collected in “ReducedModified Media” and “Human Media” were generated for retroviraltransduction of primary cell cultures.

Reduced Modified Media

DMEM:F12 supplemented with the components listed below.

-   -   Human Serum Albumin 0.03%.    -   Transferrin, Human 100 μg/ml.    -   Putrescine Dihydrochloride 16.2 μg/ml.    -   Insulin, Human recombinant 5 μg/ml.    -   Progesterone 60 ng/ml.    -   L-Glutamine 2 mM.    -   Sodium Selenite (selenium) 40 ng/ml.    -   Plus basic Fibroblast Growth Factor (10 ng/ml) and epidermal        growth factor (20 ng/ml) for cell expansion.        Cell Line Derivation

CTXOEO3 was derived under Quality Assured conditions suitable forprogressing designated lines for clinical use. As source material, humanneural stem cells were isolated post mortem from the cortex of a 12-weekgestation foetus (GS031) by enzymatic digestion with trypsin incombination with mechanical trituration. Once established in culturefoetal neural cells were infected with amphotropic retrovirus encodingthe c-MycER^(TAMTAM) oncogene, and a range of clonal and mixedpopulation cell lines isolated.

All lines in this series were derived on laminin coated culture-ware andusing serum free Reduced Modified Media (RMM) comprising DMEM: F12 basemedia, plus designated supplements as described above and growth factors(bFGF and EGF, as described above).

Growth Characteristics

Under routine culture conditions cells are expanded from frozen stocks,usually 2-4 million cells in T180 culture flasks. After several mediachanges the cells are passaged when sub-confluent. From process records,population-doubling times for CTXOE03 have been estimated at 3-4 days.This doubling time is slower than for log phase growth and also includescell loss during the process of subculture.

As a more representative assessment of log phase growth for CTX0E03, acell proliferation assay was set up using the Cyquant fluorescent dye(Molecular Probes, Invitrogen Inc.). Cell number is measured using aTecan Magellan fluorescence plate reader; ex 480 nm; em 520 nm.

CTX0E03 cells were passaged, resuspended in RMM plus growth factors andseeded on laminin coated 96 well strip-well plates at 4000 cells/well. Atime course study was carried out by removing strips from the plate,removing the media and freezing the cells at −70° C. The media wasreplaced on the remaining strips (RMM/HM+GFs+4OHT). Each subsequent daymedia was removed from the next two strips on the plate and frozen at−70° C. At the end of the time course all the frozen strips were putback together on the plate and analysed with the Cyquant assay. Brieflycells are lysed in lysis buffer then Cyquant reagent added and placed indark for 5 min. A 150 ul sample of each well was then transferred toblack, Optilux plates for reading on a Tecan Magellan plate reader. Datawas exported to a spreadsheet for numerical averaging and furtherexported to GraphPad Prism for analysis. The results are shown in FIG.1.

Conditionality of C-myc Growth Promoting Protein

Several studies have been undertaken to consistently demonstrate4-hydroxytamoxifen conditionality of the c-mycER. Cell growth in thepresence or absence of growth factors was enhanced by the application of4-hydroxy-tamoxifen in the culture media. This effect was not mimickedby beta-estradiol indicating the selective mutation of the estrogenreceptor is functionally maintained. The results are shown in FIG. 2.

Phenotype.

The phenotype of the CTX0E03 has been profiled using immunocytochemistryto stain for the neural stem cell marker nestin and to stain for maturemarkers of differentiation, beta-III tubulin (neuronal) and GFAP(astrocytic).

An assay was established to profile CTXOE3 phenotype in the presence andabsence of growth factors plus 4-OHT. Cells were passaged from routineculture and seeded in 96 well tissue culture plates at 4000 cells/well.Two plates were set up. After 3 days one plate of cells in growth mediaplus 4-OHT was fixed in 4% paraformaldehyde whilst growth factors and4OHT were removed in the other plate. After a further 5 days withoutgrowth factors the second “differentiated” plate of cells was fixed inthe same manner. This assay was repeated several times.

Differentiation ICC Images

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature,washed with PBS and permeabilized with 0.1% Triton X100/PBS for 15minutes. Non-specific binding was then blocked with 10% Normal GoatSerum (NGS) in PBS for 1 hour at room temperature. Cells were thenprobed with antibodies to Nestin (1:200 Chemicon MAB 5326), Beta-IIITubulin (1:500; Sigma) and GFAP (1:5000; DAKO) at room temperatureovernight. After washing with PBS, they were then processed withfiltered Alexa Goat α Mouse 488 (1:200; Molecular Probes) and Alexa Goatα Rabbit 568 (1:2500; Molecular Probes) dissolved in 1% NGS/PBS for 1hour at room temperature. They were then washed with PBS andcounterstained with 1 μg/ml Hoechst 33342 (Sigma) for 2 mins beforebeing analysed on a fluorescent microscope.

The results showed down-regulation of nestin and up-regulation of theneuronal marker beta-III tubulin indicating that in the absence ofgrowth factors and 4-OHT for 5 days the cells have differentiated to amore mature neuronal phenotype. There was a background of GFAP(astrocytic) staining which stayed more or less constant.

MHC Class I & II

The expression of MHC antigens on cell lines may contribute tosusceptibility of cells to rejection by the host. We have profiled theexpression of MHC class I and II on control and differentiated (growthfactor withdrawal for 14 days) CTX0E03 cells. Cells were fixed in 4%paraformaldehyde at room temperature then in methanol at −20° C. for 20mins. After blocking with normal goat serum primary antibody was addedat 1:100 dilution; [Class I, HLA-ABC #7855 (AbCam) or Class II, HLA-DR#7856 (AbCam)]. CTX 0E03 had no class II expression in contrast to acontrol cell line 0E33. Similarly CTX0E03 was negative for Class Iexpression in both undifferentiated and differentiated cells.

Genetic Stability

G-banding Karyotype Analysis

Karyotyping was carried out as follows:

From T25 flask cultures, 70-80% confluent cells were washed, stainedwith Bromodeoxyuridine, treated with Colcimid and subjected to hypotoniclysis. Samples were then fixed in methanol: glacial acetic acid [3:1]and stored at −20° C. Samples were then analysed and shown to have anormal karyotype. G-Banding analysis was conducted approximately everyten passages from Passage 5 to Passage 40, yielding normal diploidchromosomes with no abnormalities detected.Clonality/Southern Blot for CTX-OE-03

Southern transfer and hybridisation can be used to study the clonalityand integration of the retrovirally-itransduced genetic construct withinthe genome of cell lines by mapping with a probe specific for theconstruct. Genomic DNA (GDNA) is first digested with restriction enzymeand the resulting fragments are separated according to size by agarosegel electrophoresis. The DNA is then denatured in situ and transferredfrom the gel to a solid support (nitrocellulose membrane). The DNAattached to the membrane is hybridised to a ³²P labelled DNA probeagainst a specific portion of the construct and bands complementary tothe probe are located by autoradiography. If the cell lines are clonaland with only a single integration site, only one band of specific sizewill be present, if not clonal then two or more bands corresponding todifferent integration sites will be present.

Results

The Southern blot for CTX-OE-03 shows this cell line is clonal with asingle band present at 6 kb.

Genome Integration Site

Integration of retroviral DNA into the target cell genome is primarily arandom event. Inverse PCR has been widely used to detect the integrationsite of retroviral transgenes in many cell lines (Schmidt et al., ANNALSOF THE NEW YORK ACADEMY OF SCIENCES. 2001. 938:146-155). It is also apowerful tool to identify clonality. Here inverse PCR was used toidentify the pLNC-myc-ER^(TAM) integration site in CTX-0E03 line.

Genomic DNA was isolated from CTX-0E03 (5×10⁶ cells), and digestedover-night with Hind III. The digested DNA was ligated by using T4 DNAligase to form circular DNA. Two pairs of primers targeting a specificregion of the mycER gene were used to PCR the whole circular DNA. Usingthis approach the flanking sequence of the c-MycER insertion isrecovered. A ˜6 kb fragment was detected in 1.2% agrose gel thatcontains the flanking regions corresponding to the genome integrationsite.

Safety

Maternal serology indicate the donor to be free from the followinginfections;

HIV 1&2 Abs Not detected CMV IgM Not detected Hep C Ab Not detected HepB surface Ag Not detected Toxoplasma IgG Not detected Anti-HTLV-I/II EIAnegative Anti-HTLV-I GPA negative VDRL Slide Test./TPHA test SyphilisnegativePositive serology was found as follows.

CMV IgG detected

(II) Derivation and Provenance of the C-MycER^(TAM) Immortalised CellLine STR0C05

Summary

A c-MycER^(TAM) transduced-neural stem cell line was derived from 12week fetal striatum. The line is maintained on laminin coated cultureflasks using defined serum free “Human Media” in the presence of bFGF,EGF and 4-hydroxy tamoxifen. In routine culture the cell line has adoubling time of 3-4 days although in short term culture a doubling timeof 20-30 h was seen.

In growth medium the cells are nestin-positive, beta-IIItubulin-negative with a low percentage of GFAP positive cells. Followingdifferentiation for 7 days there is down regulation of nestin withlow-level expression of beta III tubulin and strong expression of GFAPsuggesting that the cell line becomes predominantly astrocytic.

This cell line is genetically normal, male XY, and stable over 50population doublings.

Introduction

The lines described here were derived under Quality Assured conditionssuitable for progressing designated lines for clinical use. As sourcematerial, human neural stem cells were isolated post mortem from thestriatum of a 12-week gestation fetus GS006 by enzymatic digestion withtrypsin in combination with mechanical trituration. Once established inculture these primary neural cells were transformed by retroviraltransduction with the c-MycER^(TAM) oncogene (as described for theCTXOEO3 cell line above) and a range of clonal and mixed population celllines isolated. All lines in this series were derived on laminin coatedculture-ware and using Human Media (HM); DMEM:F12 plus designatedsupplements as described below.

Human Media (HM)

DMEM:F12 supplemented with the components listed below.

Human Serum Albumin 0.03%. Transferrin, Human 100 μg/ml. PutrescineDihydrochloride 16.2 μg/ml. Insulin, Human recombinant 5 μg/ml.L-Thyroxine (T4) 400 ng/ml. Tri-Iodo-Thyronine (T3) 337 ng/ml.Progesterone 60 ng/ml. L-Glutamine 2 mM. Sodium Selenite (selenium) 40ng/ml. Heparin, sodium salt 10 Units/ml. Corticosterone 40 ng/ml.Plus basic Fibroblast Growth Factor (10 ng/ml) and epidermal growthfactor (20 ng/ml) for cell expansion.Growth Characteristics

Under routine culture conditions cells are expanded from frozen stocks,usually 2-4 million cells in T180 culture flasks After several mediachanges the cells are passaged when confluent. From process records,population doubling times for STR0C05 have been estimated at 3-4 days asshown on the graph below. This doubling time is slower than for logphase growth and also includes cell loss during the passaging.

As a more representative assessment of log phase growth for STR0C05, acell proliferation assay was set up using the Cyquant fluorescent dye(Molecular Probes). Cell number is measured using a Tecan Magellanfluorescence plate reader; ex. 480 nm; em 520 nm.

STR0C05 cells were passaged, resuspended in HM plus growth factors andseeded on laminin coated 96 well strip-well plates at 5000 cells/well. Atime course study was carried out by removing strips from the plate on adaily basis, n=16 wells per time point, removing the media and freezingthe cells at −70° C.

At the end of the time course all the frozen strips were put backtogether on the plate and analysed with the Cyquant assay. Briefly cellsare lysed in lysis buffer then Cyquant reagent added and placed in darkfor 5 minutes. A 150 ul sample of each well was then transferred toblack, Optilux plates for reading on a Tecan Magellan plate reader. Datawas exported to an Excel spreadsheet for numerical averaging and furtherexported to GraphPad Prism for analysis.

The results showed that the cells grew steadily over 7 days with anestimated doubling time of 20-30 hours.

Phenotype

The phenotype of the STR0C05 has been profiled using immunocytochemistryto stain for the neural stem cell marker nestin and to stain for maturemarkers of differentiation, beta-III tubulin (neuronal) and GFAP(astrocytic).

STR0C05 phenotype was determined in the presence and absence of growthfactors plus 4-OHT. Cells were originally sourced from STR0C05 workingstock. Cells were passaged and seeded in 96 well plates.

Cells were fixed in 4% paraformaldehyde for 15 minutes at roomtemperature, washed with PBS and permeabilisd with 0.1% Triton X100/PBSfor 15 minutes. Non-specific binding was then blocked with 10% NormalGoat Serum (NGS) in PBS for 1 hour at room temperature. Cells were thenprobed with antibodies to Nestin (1:200, Chemicon), Beta-III Tubulin(1:500; Sigma) and GFAP (1:5000; DAKO) at room temperature overnight.After washing with PBS, they were then processed with filtered AlexaGoat a Mouse 488 (1:200; Molecular Probes) and Alexa Goat α Rabbit 568(1:2500; Molecular Probes) dissolved in 1% NGS/PBS for 1 hour at roomtemperature. They were then washed with PBS and counterstained withHoechst 33342 (Sigma) for 2 minutes before being analysed on afluorescent microscope.

Removal of growth factors and 4-OHT from the medium induces amorphological and phenotypic change in the cells that is accompanied bydown regulation of nestin. Specifically a small proportion of the cellsbecome positive for the neuronal marker beta-III tubulin and acquire aneuronal morphology with rounded cell bodies extending intodendritic/axonal outgrowths. The more dominant phenotypic change howeveris the up-regulation of GFAP suggesting a predominance of an astrocyticlineage.

Clonality

Southern Blot for STR0C05

To date in two separate experiments there is no evidence of probehybridisation in contrast to clear bands seen with other cell lines.

(III) Derivation and Provenance of the C-mycER^(TAM) Immortalised CellLine HPC0A07

Summary

An immortalised neural stem cell line was derived from 12-week fetalhippocampus. The line is maintained on laminin coated culture flasksusing defined serum free “Reduced Modified Media” in the presence ofbFGF, EGF and 4-hydroxytamoxifen. In routine culture the cell line has adoubling time of around 2 days; in short term culture a doubling time of20-30 hours is also seen.

In growth medium the cells are nestin positive beta-III tubulin negativewith a low percentage of GFAP positive cells. Following differentiationfor 7 days there is up-regulation of beta III tubulin expression andacquisition of a neuronal morphology. A low level background of GFAPexpression is maintained following differentiation.

Following differentiation there is up-regulation of MHC class IIantigens but not Class I.

Molecular phenotyping by RT-PCR has confirmed the cells to be mycERpositive and nestin positive. In addition a range of neural developmentgenes have been identified as present.

This cell line is karyotypically normal, female XX, at passage 8 (over20 population doublings). Later passage data is currently beingprocessed.

Maternal serology indicates the donor to be free from major infections.

Introduction

As source material, human neural stem cells were isolated post mortemfrom the hippocampus of a 12-week gestation foetus by enzymaticdigestion with trypsin in combination with mechanical trituration. Onceestablished in culture foetal neural cells were transformed byretroviral transduction with the mycER^(TAM) oncogene, and a range ofclonal and mixed population cell lines isolated.

All lines in this series were derived on laminin coated culture-ware andusing serum free media known as Reduced Modified Media (RMM) (see above)for composition comprising DMEM:F12 base media, plus designatedsupplements.

Growth Characteristics

Under routine culture conditions cells are expanded from frozen stocks,usually 2-4 million cells in T180 culture flasks. After several mediachanges the cells are passaged when confluent. From process records,population doubling times for HPC0A07 have been estimated at 3-4 days.This doubling time is slower than for log phase growth and also includescell loss during the passaging

Cell Proliferation Assay

As a more representative assessment of log phase growth for HPC0A07, acell proliferation assay was set up using the Cyquant fluorescent dye(Molecular Probes). Cell number is measured using a Tecan Magellanfluorescence plate reader; ex.480 nm; em 520 nm.

HPC0A07 cells were passaged, resuspended in RMM plus growth factors andseeded on laminin coated 96 well strip-well plates at 4000 cells/well. Atime course study was carried out by removing strips from the plate,removing the media and freezing the cells at −70° C. The media wasreplaced on the remaining strips (RMM/HM+GFs+4OHT). Each subsequent daymedia was removed from the next two strips on the plate and frozen at−70° C.

At the end of the time course all the frozen strips were put backtogether on the plate and analysed with the cyquant assay. Briefly cellsare lysed in lysis buffer then Cyquant reagent added and placed in darkfor 5 minutes. A 150 ul sample of each well was then transferred toblack, Optilux plates for reading on a Tecan Magellan plate reader.

The cells expanded over 4-5 days with a doubling time of 1-2 days (asshown in FIG. 3).

Phenotype

The phenotype of the HPC0A07 has been profiled using immunocytochemistryto stain for the neural stem cell marker nestin and to stain for maturemarkers of differentiation, beta-III tubulin (neuronal) and GFAP(astrocytic).

An experiment was carried out to profile HPC0A07 phenotype in thepresence and absence of growth factors plus 4-OHT. Control(undifferentiated) cells and cells grown for 7 days in the absence ofgrowth factors and 4-OHT were fixed in 4% paraformaldehyde and subjectedto immunocytochemistry as described.

Cells were fixed in 4% paraformaldehyde for 15 minutes at roomtemperature, washed with PBS and permeabilizd with 0.1% Triton X100/PBSfor 15 minutes. Non-specific binding was then blocked with 10% NormalGoat Serum (NGS) in PBS for 1 hour at room temperature. Cells were thenprobed with Beta-III Tubulin (1:500; Sigma) and GFAP (1:5000; DAKO) atroom temperature overnight. After washing with PBS, they were thenprocessed with filtered Alexa Goat α Mouse 488 (1:200; Molecular Probes)and Alexa Goat α Rabbit 568 (1:2500; Molecular Probes) dissolved in 1%NGS/PBS for 1 hour at room temperature. They were then washed with PBSand counterstained with Hoechst 33342 (Sigma) for 2 minutes before beinganalysed on a fluorescent microscope.

The images show strong up-regulation of the neuronal marker beta-IIItubulin accompanied by morphological changes consistent with neurones.There is a low level of astrocytic cells in both control anddifferentiated cells as indicated by the GFAP staining.

MHC Class I & II

Cells were set up in laminin coated 96 well plates and expanded in RMMplus growth factors plus 4-OHT. Cells were differentiated for 7 or 14days by removing growth factors and 4OHT from the medium.

MHC class I is not expressed in control or differentiated cells after 7or 14 days. By contrast MHC II is strongly expressed in differentiatedcells especially at 14 days. Undifferentiated cells show no MHCIIexpression.

Genetic Stability

Karyotyping Analysis

The cells were shown to have a normal female karyotype.

Clonality

Southern blot for HPC0A07 shows a single band at 5 kb suggesting thatthis cell line is clonal.

Safety

Maternal serology from GS011 indicates the donor to be free from thefollowing infections;

HIV 1&2 Abs Not detected CMV IgG Not detected CMV IgM Not detected Hep CAb Not detected Hep B surface Ag Not detected Toxoplasma IgG Notdetected Anti-HTLV-I/II EIA negative Anti-HTLV-I GPA negative VDRL SlideTest/TPHA test Syphilis negativeTherapeutic Use of CellsCTXOEO3 Stem Cell Line Reduces Deficits After MCAo Stroke in Rats.

The aim of this study was to test the efficacy of human neural stem celllines developed by this invention in promoting functional recovery aftermiddle cerebral artery occlusion in rat, a validated model of embolicstroke in humans.

Two human cell lines, CTXOE03 (n=10) and a cell line with similarphenotype derived from the striatum, STROB05 (n=9), were implanted intocortex and striatum (bilaterally) 3 to 4 weeks after stroke induced by70 minutes of intraluminal right middle cerebral artery occlusion. Therewere two vehicle grafted control groups: stroke (n=7) and sham operated(n=11). Six to twelve weeks after grafting, all four groups were testedfor bilateral asymmetry, vibrissae-elicited forelimb placing, rotationbias and a spatial learning task before histological investigation oflesion volume, cell distribution and differentiation.

Rats with cell line grafts displayed reduced bilateral asymmetry andamphetamine induced rotation when compared to the stroke controlanimals. The STROB05 group also displayed reduced dysfunction in theforelimb placing test compared to the stroke controls. Theseimprovements did not reach sham control levels. Neither of the cell linegrafted groups showed reduced dysfunction in the spatial learning taskor differences in lesion volume compared to the stroke alone group.STROB05 grafts survived well, but grafted cells were seen in a smallernumber of the rats in the CTXOE03 group. Neither cell line showedneuronal differentiation at 14 weeks after grafting. In conclusion, bothof the grafted cell lines promoted behavioural recovery over the courseof the study.

Middle cerebral artery occlusion (MCAo) produces behavioural deficitsthat are evident using neurological, sensori-motor, and spatial memorytests. There is spontaneous resolution of many deficits in animals by 30days after occlusion paralleling recovery seen in patients over monthsof recovery after stroke. We used tests in this study that are robust inidentifying long-term deficits, ensuring that improvements reflect truerecovery, not reduced stroke damage or accelerated rehabilitation.

Materials and Methods

MCAo

Male Sprague-Dawley rats (Charles Rivers) were group housed (12:12 hourslight dark) for 7 days prior to occlusion, with food available adlibitum. Procedures complied with the UK Animals (Scientific) ProceduresAct (1986) and the Ethical Review Process of ReNeuron Ltd.

Animals were prepared for surgery (320-370 g) and subjected to 70minutes of middle cerebral artery occlusion using a 3-0 prolene filamentcoated with silicone (Halford's UK). Halothane (in 70% NO2/30% O₂)anaesthesia was used for insertion and removal of the filament, withtemperatures held at 37±1° C. by rectal probe and heating pad. Once thefilament was inserted (˜20 mm), animals were allowed to recover fromanaesthesia. At 60 minutes into occlusion, animals were evaluated forbehavioural dysfunction (forelimb flexion and contralateral circlingbehaviour). Animals that did not demonstrate dysfunction were removedfrom the study. At the end of the occlusion period, the animals werere-anaesthetized and the filament partially withdrawn to clear the baseof the MCA. The wound was closed, saline administered, and the animalsplaced in a heated chamber for 2 hours. Animals were put intopost-operative care. Sham MCAo animals underwent anaesthesia andfilament placement of 5-6 mm.

For 2 days after MCAo, all animals had rehydration therapy (Duphalyte[Fort Dodge]) in saline, 5 mls 50:50 mixture), were scored forneurological dysfunction and weighed (further rehydration therapy wasadministered as necessary with continued scoring and weighing). Oncepre-occlusion body weight was reached, animals were tested on the taperemoval and rotameter tasks (see below) to provide baseline scores forthe test battery and to allow for balancing experimental groups. Ratswere assigned to either sham, vehicle infusion of N-acetyl-L-cysteine(NAC), STROB05 grafting or CTXOE03 grafting.

The STROB05 and CTXOE03 cell lines were harvested and suspended at aconcentration of ˜50,000 cells/ul in NAC (Sigma). Cells were preparedtwice during the days of grafting. Cell viability was recorded both atthe beginning of transplantation and after grafting via trypan blueexclusion (Sigma). Cell viability was high (over 85%) before and aftergrafting sessions. Control MCAO and sham animals both had infusions ofthe grafting vehicle, NAC (Sigma) in Hanks' balanced salt solution(Gibco).

At 3-4 weeks after occlusion, animals were injected with cyclosporin A(CSA)(Sandimmun, 10 mg/kg SC: Sandoz) in Cremaphore el (Sigma), thengrafted 24 hours later. Animals were pre-anaesthetized with medetomidinehydrochloride (Domitor, 0.5 mg/kg, i.p. Pfizer), then anaesthetized withketamine hydrochloride (Vetalar 80 mg/kg, i.p., Pharmacia Upjohn). Afterpreparation, animals were placed into stereotaxic frames, the scalpincised, skull exposed and bregma located. Grafts were deposited using aflat tipped 10 μl Hamilton syringe at 4 sites, ipsilateral andcontralateral to the occluded hemisphere at the following coordinatesrelative to bregma: (+0.7 mm AP, ±3.0 mmLat, −5.5, −2.0 mm Vert; −0.3mmAP, ±0.35 mm Lat, −5.5, −2.0 mm Vert). Cells were injected at a rateof 1 ul/minute and the syringe kept in place for another minute toreduce reflux. Once all cells were injected (400,000/animal), the woundswere sutured shut, the animal given saline rehydration, andanti-inflammatory treatment; (methylprednisilone; medrone 20 mg/kg sc,Pharmacia Upjohn) and CSA. Anaesthesia was reversed with atipamezolehydrochloride (antisedan, 0.1 mg/kg, Pfizer) and analgesic was providedwith bupranorphine hydrochloride (vetergesic, 0.1 mg/kg, Alstoe AnimalHealth). Once animals had recovered fully from anaesthesia, they wereplaced into post-operative care, including rehydration therapy for 2days after surgery. Animals received post-operative immunosuppresiontreatment with medrone daily for 20 days, and CSA thrice weekly over thecourse of the study.

At 6 weeks after grafting, animals began the sensori-motor test battery.Animals had 1 forelimb placing test per week with 3 trials per session,2 sticky tape tests with 2 trials per session, and 1 half hour rotametertest per week, alternating saline and amphetamine trials. At 12 weekspost-grafting, the sensori-motor testing ended and the animals weretested for acquisition in the Morris water maze for 10 working days.

Vibrissae-elicited Forelimb Placing Test

Animals were held with one forelimb free and moved toward a tabletop.When the vibrissae brush the table, the ipsilateral paw is placed ontothe tabletop. The number of appropriate limb placements after 3 brushesper side was recorded.

Bilateral Asymmetry

The bilateral asymmetry test of sensorimotor dysfunction measured thedisparity in time taken to contact and remove sticky tape strips (˜1×6cm) wrapped around the affected and unaffected forepaws for 300 seconds

Rotameter

The rotameter (TSE GmbH) measured motor rotation asymmetry in responseto amphetamine (2.5 mg/kg sc, Sigma) or saline administration. Rotationbias was calculated, as the number of anti-clockwise turns divided bythe total number of turns in both directions.

Histological Evaluation

Animals were overdosed with pentobarbitone (Animalcare), flushedtranscardially with heparinized saline, and perfused with 4%paraformaldehyde in 0.2 mol/L PBS. Brains were cryoprotected by 30%sucrose and cut by sliding cryo-microtome (Frigomobile:Leica) into 50 μmsections. Lesion volume was calculated in every 20^(th) section bySimpson's rule with the use of digital microscope images analysed byImage Pro Plus (Media Cybernetics). Transplanted cells were identifiedby the use of antibodies raised against human nuclear protein(HuNuc—Chemicon Inc). Differentiation of transplanted cells wasdetermined by colocalizing phenotypic markers, neurofilament and KI-67with the human protein. Primary antibodies were incubated overnightbefore the fluorescent anti-mouse or anti-rabbit secondary antibody wasapplied for 1 hour.

Statistical Analyses

Results for the bilateral asymmetry tests were analysed by 2-way ANOVA(Prism) with groups as the between-subjects factor and trials/days asthe within-subjects factor. t-tests were used to analyse prelesionversus postlesion performance in the rotameter test. One-way ANOVAs wereused for group differences on the rotameter, whiskers, and lesionvolumes. The Bonferroni post hoc test was used to compare groups.

Results

Occluded animals showed gross neurological dysfunction for the first 7to 14 days after occlusion. This dysfunction was accompanied with mildweight loss (<30%). Dysfunction and weight loss were usually resolvedwithin 14 days after occlusion. Post-hoc analysis determined that weightloss at 7 days post occlusion correlated to final lesion volume whenanalysing all animals in the study (r=−0.705, p<0.0001).

Vibrissae-elicited Forelimb Placing Test

There were significant differences in the number of limb placementsbetween the affected and non-affected limbs in all three of the occludedgroups (p<0.0001). There was no difference in limb placements within thesham group. There was a significant difference in placements of theaffected limb between the sham group and the occluded groups (p<0.001all groups). There was also a significant difference in placements ofthe affected limb between the STROB05 grafted and the stroke control(p<0.01) and CTXOE03 (p<0.001) grafted groups. The STROB05 group did notreach the levels of placements seen in the sham group for the affectedlimb.

Bilateral Asymmetry Task

Contact; There were significant differences in the time to contact thetape on the affected (left) paw with the stroke control treated animalstaking longer than the sham, STROB05 and CTXOE03 grafted groups (p<0.01all groups, ANOVA). There were also significant differences betweengroups with the sham animals taking longer than the stroke controls andCTXOE03 groups in contacting the tape on the non-affected paw (p<0.05,ANOVA).

Removal; There were significant differences between groups with the NACtreated animals taking longer than the sham, STROB05, and CTXOE03grafted groups in removing the tape from the affected (left) paw(p<0.001, all groups ANOVA). There were significant differences betweengroups with the sham animals taking longer than the NAC, STROB05, andCTXOE03 grafted groups in removing the tape from the non-affected(right) paw (p<0.0001 all groups, 2-way ANOVA).

Rotameter

Saline: Asymmetry of spontaneous rotations following salineadministration was significantly lower in the CTXOE03 and sham groupswhen compared to the NAC treated group after grafting (p<0.05). Therewas a significant reduction in the bias of spontaneous rotations aftergrafting when compared to pre-grafting performance in the STROB05(p<0.01) and CTXOE 03 (p<0.05) groups.

Amphetamine: Asymmetry of amphetamine induced rotations, ipsilateral tothe lesion was reduced in the cell line grafted and sham animals whencompared to the occluded NAC treated animals after grafting (p<0.001).There was still a significant difference between the sham and cell linegrafted groups with the grafted groups having a higher percentage ofcounter-clockwise rotations (p<0.01). There was a significant reductionin amphetamine-induced asymmetry after grafting when compared topre-grafting performance in the STROB05 (p<0.05) and CTXOE03 (p<0.01)groups.

Lesion Volumes; There were significant differences in the volume ofdamaged tissue between the sham (24.0

≡˜5.1 mm³) and the NAC (220.6

≡˜32.5 mm³), STROB05 (250.0

≡˜37.0 mm³) and CTXOEO3 (214.2

≡˜36.5 mm³) groups (p<0.001 all groups, ANOVA), with no differencesbetween the NAC and grafted groups. Damage typically occurred throughoutthe sensory and motor cortices and striatum. Thalamic atrophy indicatedsecondary degeneration, however, no gross damage to the hippocampus wasapparent.

Immunohistochemistry

Human cells were identified in both the STROB05 and CTXOE03 graftedgroups. There was good survival in most of the STROB05 grafted animals(7/9) but CTXOE03 animals had good survival in only 2 of 9 animals.Neither cell line demonstrated differentiation into neurons in any ofthe animals at long-term survival. The STROB05 cell had someco-localization of human nuclear and KI-67 protein expression (a markerfor cell division) in the areas of cavitation outside the parenchymasuggesting that local cues may have promoted cell division. There wasgood migration in one of the CTXOE03 grafted animals with cellsmigrating throughout the sections.

STROCO5 and CTXIE03 Stem Cell Lines Reduce Deficits After QuinolinicAcid Lesions of the Sriatum (Modelling Huntington's Disease) in Rats

The study aimed to assess effects of human MycER^(TAM) cell linesSTROCO5 nd CTXOEO3 in rats with deficits induced by unilateral (leftside) quinolinic acid striatal lesions, as a partial model forHuntington's disease (HD).

Quinolinic acid produced selective damage to DARPP 32 positive neuronsin the striatum, which was uniform and across groups spared other celltypes. These discrete lesions did not result in amphetamine-inducedrotation bias or deficits in spatial learning. However deficits wereseen in tests of sensorimotor (sticky tape removal times) and motorfunction (pellet retrieval on the staircase) especially with the rightpaw. There was a marked lesion effect in elicited reflex responses shownin the body swing and whiskers tests. The STROCO5 grafted group showedsignificant improvement relative to lesion-only animals in pelletretrieval on the staircase test, in body swing bias and paw placementafter whisker stimulation. The CTXOEO3 grafted group showed similarimprovements in performance in the body swing bias and paw placementafter whisker stimulation. Grafts were visualised long term in about 50%of the grafted rats. These results indicate a potential for repair of arange of motor responses in rats with HD-like loss of medium spinystriatal output neurons.

Materials and Methods

Male Sprague-Dawley rats (Charles Rivers) were group housed (12:12 hourslight dark) for 7 days prior to surgery, with food available ad libitum.Procedures complied with the UK Animals (Scientific) Procedures Act(1986) and the Ethical Review Process of ReNeuron Ltd.

Animals were prepared for surgery (mean weight 379 g). For lesions, therats were anaesthetised with ketamine (Ketalar, 80 mg/kg, Pharmacia andUpjohn Ltd. UK) and medetomidine (Domitor 0.5 mg/kg, Pfizer, UK)reversed after surgery by Atipamezole (Antisedan; Pfizer, UK) and placedin a stereotaxic frame (Kopf, Tujunga Calif.). A homeothermic blanketwas used to maintain body heat. Holes were drilled in the skull to allowinsertion of a cannula attached by tubing to a 10 ul Hamilton Syringedriven by a pump (Harvard Instruments). The following coordinates (mm)were used, for a left-sided lesion with the skull held in flat position(3.5 mm below the inter aural line).

(1) AP=0.0; L=+3.5; V=−4.5 and (2) AP=+1.2; L=+2.8; V=−4.5

0.08 M quinolininic acid (Sigma, UK) was prepared by weighing out 13.368mg of quinolinic acid ((2,3-Pyridinedicarboxylic Acid, Sigma UK), adding0.5 ml of PBS and 10-20 ul of 1 M sodium hydroxide solution, andsonicating until dissolved. PH was checked and adjusted if required, andthe solution made up to 1 ml with PBS. 1.0 ul of quinolinic acid wasinfused over 2 minutes. The cannula was left in place for a further 2minutes, to allow diffusion of the toxin. Surgery took place over twoweeks.

Surgery—Grafting: Rats were anaesthetised and placed in the frame, andthe skull sites prepared as for lesioning. A 10 ul flat-tipped Hamiltonsyringe into the lesion sites at the following coordinates deliveredcell suspensions:

1) AP=0.0; L=+3.6; V=−5.5, −4.5 and (2) AP=+1.2; L=+2.8; V=−5.5, −4.5.

2 deposits of 3 ul at a density of 50,000 cells/uil were delivered ateach site (i.e. 6 il=300,000 cells/rat) over 3 minutes. The syringe waslowered to the deepest point first and 1.5 ul cells dispensed, beforeraising the needle to the upper site to deliver the remaining 1.5 ul ofsuspension. The syringe was slowly withdrawn after waiting for 2 minutesdispersal. resh draws of suspension were taken for each site. Cells werefreshly prepared and delivered twice a day (am, pm) to reducedeterioration on the bench.

All rats received immunosuppressive treatments consisting of cyclosporinA (10 mg/kg sc) in Cremophor EL (CSA/CEL) in a ratio of 1:4, startingthe day before surgery and continuing for 3 days/week until perfusion.In addition rats were injected daily (20 mg sc) with n-methylprednisalone (Solumedron) for 2 weeks, starting on the day of surgery.

Final Group sizes were: Lesion and sham graft, N=7, Control, N=9,STROCO5, N=9, CTXOEO3, N=12.

At 6 weeks after grafting, the rats commenced behavioural testing.

Reflex Motor Response Testing:

Body Swing Test (BST). Rats were placed in an open cage facing theexperimenter and lifted by the base of the tail above floor level for 10seconds. Twists defined as distinct kicks in which hindlimbs crossed themidline to the animals' left or right were recorded. Preferably twoexperimenters were used, one to hold and one to record. This providedinter-experimenter reliability. If the rat twisted first towards thelesioned side (left in this case) a score of 1-3 was recorded for theintensity of the swing. A contralateral swing was recorded as zeroscore. Inter-rater reliability for scores was relatively low, so thisqualitative measure was not used in the final analysis of results, whichused % left swings, to measure bias.

Each session (block) consisted of 3 10-second lifts. One session wasgiven in the week after lesion surgery to assess the lesion, and 6blocks were given at weekly intervals over weeks 6-12 after grafting toassess graft effects.

Paw placement in response to vibrissae stimulation.

Rats were held with one forepaw free and the other restrained, andwhiskers adjacent to the free paw gently brushed against the side of atable. The rat immediately reached to touch the table. However rats withunilateral damage often failed to place the paw contralateral to thedamage on the tabletop. Each session consisted of three trials on eachside, in alternation. Rats were scored 1 for reaching and 0 for failureto respond within a 5 second period. Four sessions were given at the endof behavioural testing, weeks 9-10 after grafting.

Sensorimotor Dysfunction Testing:

Staircase Test:

The apparatus consists of an entry chamber giving access to a platformflanked by two seven-step staircases, with a hollow for pellets on eachstep. Rats climb onto the platform to reach pellets (2 Coco Pops;Kellogs Ltd.) on each step. A narrow lip on the edge of the platformprevented rats from scraping up the pellets, for successful retrievalpellets must be grasped and lifted to the mouth, using the right andleft forelimb for each side. Rats were given two 5-minute trials persession. Rats were trained before lesioning (2 sessions), tested afterlesioning (2 sessions) and after transplantation (4 sessions), over thesame period as BAT testing.

Histology

Perfusion and Sectioning:

Rats were transcardially perfused with 4% paraformaldehyde in 0.1 Msodium phosphate buffer (PBS, pH 7.4), brains were removed and stored infixative at 4° C. overnight, then transferred to 30% sucrose in PBS. 50μm serial sections were cut on a freezing microtome, collected insucrose in 24 well culture plates and stored at −20° C. untilprocessing.

Lesion Damage:

Lesions were quantified as the extent of loss of DARPP 32 positive cellsthroughout the left striatum; both the area of immunoreactivity and theintensity of staining were measured, using c. 8 sections running throughthe AP axis of the striatum.

Grafted Cell Survival:

Grafted cells were identified by Human Nuclear (HuNuc—Chemicon Inc)staining and counted in serial sections adjacent to those used forlesion evaluation, throughout the grafted striatum. Sections close tothe injection site were the chief focus of scrutiny.

Results

Staircase Test

More pellets were retrieved with the left than the right paw (p<0.05),across all groups. There was a substantial difference between Groups(p<0.001) since lesioned rats retrieved fewer pellets than STROCO5grafted and control groups (p<0.01).

Body Swing Test

Lesioned rats showed substantial bias in twisting to the left throughoutthe six blocks of post-transplant testing, hence Groups differed inpercentage of left swings. (p<0.001: see Fig). Hence there was asubstantial difference between groups: both the CTXOEO3 and STROCO5grafted as well as the control groups showed reduced bias (i.e. c. 50%swings to left and right), and differing significantly from lesionedrats (p<0.01). In terms of the actual numbers of swings to left andright, an interaction between Groups and side (p<0.001 underlines thebias in lesioned, but not control or grafted groups.

Paw Placement in Response to Ipsilateral Vibrissae Stimulation.

Lesioned rats failed to respond consistently to stimulation of whiskerson the right side, although reaches to the tabletop with the left pawwere fast and accurate after stimulation on the left side. Control andgrafted groups responded to stimulation on both sides. Therefore sidesdiffered significantly (p<0.001) and there was a massive interactionbetween Groups and Sides (p<0.001), as well as a substantial differencebetween Groups (p<0.001). Control and STROCO5 and CTXOEO3 grafted groupsdiffered from lesioned rats (p<0.001). However, both grafted groups wereless responsive than control rats (p<0.02) so that they still showedsome neglect.

Histological Findings

Lesion Size and Specificity

Lesions were well targeted to the striatum, and involved little tissueloss apart from the absence of DARPP 32 positive cells, and someventricular enlargement. The area of cell loss averaged 10 mm³ in bothlesion-only and grafted groups. There was also no difference in lesionvolume/intensity in grafted rats with and without surviving cells.

Graft Survival:

Surviving HuNuc positive cells were seen in 4/9 STROCO5 grafted rats and5/12 CTXOEO3 grafted rats. Good migration was seen in 75% of animalswith grafts. There was no apparent differentiation of long-termsurviving grafted cells into neurons.

HPCOAO7 and CTXOEO3 Cell Lines Restore Cognitive Performance AfterGlobal Ischaemia (4-Vessel Occlusion)

The experiment aimed to see whether grafts of two regulated humanc-MycER^(TAM) stem cell lines would be able to improve spatial learningand memory in rats that showed deficits after ischaemic hippocampaldamage induced by four vessel occlusion. Following ischaemia, rats weregrafted with cells from a hippocampal (HPC OA 07) and a cortical (CTX OE03) line. Six weeks later they were trained to find a submerged platformin a 2 m diameter circular pool, with 2 trials/day for a total of 6days, followed by a probe trial with the platform removed, to testrecall of its location. Sham grafted ischaemic rats took longer to findthe platform, swam further in searching for it, and spent less time inthe pool area where the platform was located, than non-ischaemiccontrols. The performance of grafted rats was intermediate between thatof ischaemic and non-ischaemic controls, in general not differingsignificantly from either. However improvement above the level ofischaemic rats was very close to significance in key measures such aslatency and path length, indicating a potential for reliable functionalrecovery.

Experimental Procedures

Surgery—four vessel occlusion: Rats were subjected to 4 VO byelectrocauterising (Surgitron coagulator) the vertebral arteries underhalothane anaesthesia (Merial Animal Health), and inserting ties aroundthe carotids. The next day the carotids were lifted and clamped usingserrefine aneurism clips for 20 minutes under brief halothaneanaesthesia. Loss of righting reflex was maintained after theanaesthetic was discontinued, indicating 90%+reduction of cerebral bloodflow. Controls were sham operated by cauterising the vertebral arteriesand inserting ties, but without restriction of carotid blood flow. Bodyand head temperatures were monitored by rectal and head probes, recordedeach minute and maintained at 37±2° C. by a homeothermic blanket(Harvard Apparatus) and overhead lamp.

Surgery—grafting: Rats were anaesthetised with ketamine (Ketalar, 80mg/kg, Pharmacia and Upjohn Ltd. UK) and medetomidine (Domitor 0.5mg/kg, Pfizer, UK) reversed after surgery by Atipamezole (Antisedan, 20mg/kg, Pfizer, UK) and placed in a stereotaxic frame (Kopf, Tujunga CA).Homeothermic blankets (Harvard Apparatus) were used to maintain bodyheat. Holes were drilled in the skull to allow insertion of a 10-ul flattipped Hamilton syringe. Two deposits of 2 ul at a density of 50,000cells/ul were delivered at each site.

(i.e. 8 μl=400,000 cells/rat) over 2 minutes, at the followingcoordinates:

AP: −3.3 L: ±1.3 V: −2.8 2″I/site AP: −4.2 L: ±3.4 V: −3.1

The syringe was slowly withdrawn after waiting for 2 minutes fordispersal. Fresh draws of suspension were taken for each site. Cellswere freshly prepared and delivered twice a day (am, pm) to reducedeterioration on the bench.

All rats received immunosuppressive treatments consisting of cyclosporinA 10 mg/kg sc) in Cremophor EL (CSA/CEL) in a ratio of 1:4, starting theday before surgery and continuing for 3 days/week until perfusion. Inaddition rats were injected daily (20 mg sc) with prednisolone for 2weeks, starting on the day of surgery.

Behavioural Testing for Spatial Memory Dysfunction:

Morris Water Maze, Animals were placed in a round pool (2 m) with asubmerged platform (circle of 9 cm diam.) 2 cm under the surface of thewater, with temperature held at 24±2° C. Animals were placed in one offour starting points and allowed to swim for a maximum of one minute.When animals climbed onto the platform they were allowed to remain for10 seconds before being removed. Animals not finding the platform withinone minute were placed on the platform. Animals were left on theplatform for 20 seconds and then removed. The swim path was recorded byan image analysing system (HVS Image, UK). There were 2 trials per dailysession separated by an interval of 8-10 minutes. Training was carriedout over 9 days, in blocks of 4 and 3 days. However, on the 4^(th) dayan apparatus failure resulted in unreliable data collection, so theresults were discarded. The data analysed were therefore collected over6 days counted as blocks 1-3 and 4-6, separated by a three-day interval.A probe trial was given 24 hous after the last acquisition trial,followed by a visible platform task on the next day. Three trials weregiven with the platform position marked by a cylindrical cue rising 10cm above the surface of the water.

Histology

Perfusion and Sectioning:

Rats were transcardially perfused with 4% paraformaldehyde in 0.1 Msodium phosphate buffer (PBS, pH 7.4), brains were removed and stored infixative at 4° C. overnight, then transferred to 30% sucrose in PBS. 50μm serial sections were cut on a freezing microtome, collected insucrose in 24 well culture plates and stored at −20° C. untilprocessing.

Neuronal Loss:

Cell loss was quantified at the two levels of the graft/vehicleinjection tracts, bilaterally, in NeuN labelled coronal sections acrossthe medial-lateral extension of the CA1 field, including cell body anddendritic regions. Both the area of NeuN immunoreactivity and theintensity of staining were measured.

Grafted Cell Survival:

Grafted cells were identified by Human Nuclear (HuNuc) staining andcounted in serial sections from the same regions of interest (ROI) usedto estimate cell loss, in adjacent serial sections.

Results

The experiment used 33 rats, 10 with CTXOE03 grafts, 9 with HPCOA07grafts, 8 with 4 VO and sham grafts and 6 sham-occluded and sham graftedcontrols.

Acquisition in the Water Maze

Five to six weeks after grafting, rats were trained to find a submergedplatform in the water maze, with 2 trials/day separated by an intervalof 8-10 minutes.

Latency: All groups showed a substantial linear decrease in time takento find the platform over Blocks (F lin [1,29]=46.76. p<0.001) whichlevelled off as some groups reached asymptotic performance (F quad[1,29]=5.26, p<0.025). Steeper decrease in controls relative to lesionedrats resulted in an interaction between Groups and the linear trend ofBlocks (F [3, 29]=4.07, p<0.02), and a trend towards an overalldifference between Groups (F [3,29]=2.47, p=0.08). Comparison of meansshowed that intact controls differed from lesion controls (p<0.015),whilst grafted groups performed at control level. Although graftedgroups showed a trend towards improvement relative to lesion-only rats(p=0.08, p=0.09), these differences were not significant. Graftedanimals were therefore intermediate between intact and lesionedcontrols.

Histology Findings

Ischaemic lesions: The extent of CA1 cell loss was identified at twolevels marked by injection tracts, and included dendritic and somaticfields. NeuN staining was mapped by area, and by intensity ofimmunofluorescence. Both measures indicated that ischaemic groupsretained only 20-25% of cells seen in controls. Damage was uniform andlocalised to the CA1 field, and similar across all groups.

Grafted cell survival: HuNuc labelled grafted cells were seen in 2/10rats with CTX OE 03 grafts and 6/9 rats in the HPC OA 07 group; 3 ratswith HPC grafts also showed good migration. There was no evidence fordifferentiation of grafted cells into neurons.

Host brain changes: Sprouting, as measured by NF staining in the CA1field, was significantly increased in the ischaemic control group, abovethe level of both sham operated and grafted animals. Grafts, therefore,reduced sprouting activity to control level. In contrast, hostneurogenesis in the dentate gyrus was significantly increased in ratswith HPC grafts, above levels seen in ischaemic controls. However,Ki67-IR was equivalent in lesioned controls and rats with CTX grafts, sothere was no evidence for an effect of these cells on ongoingneurogenesis at the time of perfusion.

-   -   Correlations Between Histological and Behavioural Measures

Mean latency to find the platform over Blocks 1-3,4-6, and over all 6training blocks was correlated with extent of CA1 cell loss in terms ofboth area and intensity of NeuN-IR, in each of the four experimentalgroups. Within each group there was no relationship between the extentof CA1 cell loss, and increase in time taken to find the platform, byeither measure of NeuN staining.

Similarly, the relationship between sprouting, as measured by area andintensity of neurofillament staining in the dentate gyrus, and latencyto find the platform was examined. No significant association was found.

CONCLUSIONS

The three different neural stem cell lines described in this inventionhave been shown to be stable, and scalable, sources of cells fortransplantation into patients with neurological disease. The range ofpotential therapies has been exemplified by showing functional recoveryin a range of animal models of brain disease and degeneration. Thetypical success of these cell lines in restoring function in more thanone disease model indicates that one or more of these cell lines mayrestore function in a variety of neurological diseases.

Great Britain Application Nos. 0421753.5, filed Sep. 30, 2004,0425767.1, filed Nov. 23, 2004, and 0427830.5, filed Dec. 20, 2004, areeach incorporated by reference herein in their entirety, including anyfigures, tables, nucleic acid sequences, amino acid sequences, anddrawings.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

1. A cell line deposited under ECACC Accession No. 04110301.